An Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinase (MAPK) cascade composed of YODA (YDA)–MKK4/MKK5–MPK3/MPK6 plays an essential role downstream of the ERECTA (ER)/ER-LIKE (ERL) receptor complex in regulating stomatal development in the leaf epidermis. STOMAGEN (STO), a peptide ligand produced in mesophyll cells, competes with EPIDERMAL PATTERNING FACTOR2 (EPF2) for binding ER/ERL receptors to promote stomatal formation. In this study, we found that activation of MPK3/MPK6 suppresses STO expression. Using MUTE and STO promoters that confer epidermis- and mesophyll-specific expression, respectively, we generated lines with cell-specific activation and suppression of MPK3/MPK6. The activation or suppression of MPK3/MPK6 in either epidermis or mesophyll cells is sufficient to alter stomatal differentiation. Epistatic analyses demonstrated that STO overexpression can rescue the suppression of stomatal formation conferred by the mesophyll-specific expression of the constitutively active MKK4^DD or MKK5^DD, but not by the epidermis-specific expression of these constitutively active MKKs. These data suggest that STO is downstream of MPK3/MPK6 in mesophyll cells, but upstream of MPK3/MPK6 in epidermal cells in stomatal development signaling. This function of the MPK3/MPK6 cascade allows it to coordinate plant epidermis development based on its activity in mesophyll cells during leaf development.

MAPK activation is a key event downstream of receptor-mediated signaling in plant immunity and development. While the activation of MAPK cascades is well-studied, their negative regulatory mechanisms remain less understood. Expression profiling of conditional gain-of-function MPK3/MPK6 plants identified the induction of Clade B phosphatase genes from the PP2C family, known as AP2Cs. Overexpression of any of the four AP2C members suppresses MAPK activation in seedlings treated with flg22. Additionally, these plants exhibited excessive stomatal formation and clustering, phenocopying mpk3 mpk6 mutants. In contrast, single AP2C loss-of-function mutants show no discernible phenotype, suggesting functional redundancy. Mutation of all four AP2C members leads to enhanced MPK3/MPK6 activation and accelerated cell death upon pathogen infection. Loss of MPK6 in the ap2c1/2/3/4 quadruple mutant background confirmed that MPK6 is a key target of AP2Cs in regulating HR cell death. Further investigation revealed that the MPK3/MPK6 and CPK5/CPK6, two critical immune signaling pathways, along with their downstream substrate, WRKY33, are involved in the activation of AP2C expression in response to PAMP treatment and pathogen infection. We conclude that AP2C1/2/3/4 function as MAPK phosphatases, collectively forming a negative feedback regulatory loop that fine-tunes the signaling strength and duration of MPK3/MPK6 cascade to balance plant immunity and development.